The Human
HPLC Column
Students learn about chemical separation and the
detrimental effects of drugs on the brain
T
K y l e F ra n t z
eaching complex concepts in neuroscience, biology, psychology, and chemistry
can be fun! In this active, “minds-on”
role-playing experience, students identify and explain key steps in chemical separation
by high performance liquid chromatography
(HPLC), while inferring the detrimental effects
of drugs on the brain.
Initiatives in education reform emphasize
inquiry-based active learning and realworld relevance to increase science
literacy nationwide (Cameron and
Chudler 2003). Active teaching
and learning approaches yield
rapid intellectual development (Allen and Tanner
2003; Hammer and Schifter
2001; Hofstein and Lunetta
2004) and may increase interest and motivation to learn
science (Moreno 1999). One
highly relevant topic for
adolescents is the impact of
drug abuse on the nervous
system. Out of 49,300 U.S.
secondary school students
surveyed, half of them will
have tried an illicit drug by
the time they complete high school (Johnston et al.
2005). Therefore, incorporating the topic of drug
use with neuroscience, biology, psychology, health,
chemistry, and math may increase attention and
participation in the classroom, as well as emphasize
the harmful effects of drug use and abuse.
The teaching module described in this article
should take approximately two 50-minute class
periods or one block period. Materials and expenses
are minimal. This teaching module was designed to:
introduce adolescents to
the acute neurochemical
effects of psychomotor
stimulant drugs;
◆ help students
explore two laboratory techniques
used to analyze
drug effects, in
vivo microdialysis
and chemical separation by HPLC;
◆ spur maturation of analytical reasoning skills among
adolescents; and
◆ spark enthusiasm for science.
◆
January 2007
33
FIGURE 1
Explorer Guide.
What makes you feel good? Why? How?
1. List three activities you enjoy.
2. For each of the activities listed in question 1, what benefits do you think you gain by doing them? What benefits do other
humans gain from them? Are the activities good for your health?
3. How do you think your body lets you know that you enjoy these activities?
4. What is the reward pathway and what is its role in your behavior?
5. What specific chemical is associated with reward pathways in the human brain? How does that chemical respond to
pleasurable activities?
6. How do psychomotor stimulant drugs trick humans and other animals into thinking they are experiencing pleasure?
7. How do we know the answer to question 6? List at least two ways scientists measure chemicals in the brain.
8. Is extracellular fluid itself actually extracted from the brain when using in vivo microdialysis (see Figure 2)? If not, what is? List
at least two possible analytes of interest.
9. Define laws of diffusion and explain what role they play in microdialysis.
10. What does HPLC stand for? What is the main goal in HPLC?
11. What is your role in the Human HPLC Column? What do you represent?
12. Complete the data table below, based on your in-class experiment.
Analyte
Retention time(s)
Number of analyte molecules (amount of analyte)
Average retention time(s) by type of analyte
13. Draw and label a sample chromatogram (right), based on
your own data. (Be sure to include axis labels, peak labels,
a data line, and a title.)
14. How does cocaine increase dopamine in the extracellular
fluid and in the dialysis sample?
15. What is drug tolerance? How would tolerance to the
effects of cocaine change your chromatogram? (Write
down your answer and draw another line on question
13’s graph in a different color or style, such as a dashed
line. Clearly label the new line.)
16. Consider the fact that prolonged or repeated exposure to drugs like cocaine results in long-term decreases in dopamine
activity in the brain. If high levels of extracellular dopamine are associated with activities that make humans feel
good, then how might low levels of dopamine make us feel? What does that indicate about the long-term effects of
drug abuse?
34
The Science Teacher
Providing a hook
To engage students at the beginning of the lesson, ask
them, “What makes us feel good and why?” As you brainstorm a list of potential answers on the board, have students record the list in their Explorer Guides (Figure 1).
Point out that many activities that make us feel good are
actually good for our health, as individuals or as a species
(e.g., eating healthy foods, drinking water, and nurturing).
Transition students into thinking about the brain by explaining that many activities we enjoy are associated with
the release of a chemical messenger in the brain called dopamine. Explain that the purpose of the lesson is to understand how scientists in the laboratory learn about chemicals
in the brain, and how we identify relationships between
brain chemicals and the way we and other animals behave
and feel. The lesson is also about how drugs alter those
chemicals and often compromise our ability to feel good.
Background information
One key component to this lesson plan is the National
Institute on Drug Abuse (NIDA) Slide Teaching Packet
1 (www.nida.nih.gov/pubs/Teaching) and its accompanying informative script. Download and use the packet in
class as a PowerPoint presentation or to create overhead
transparencies. Request that students take notes on this
material, which they will later use when completing the
Explorer Guide questions (Figure 1). Be sure to highlight
some specific information from the script, including
◆
◆
the reward pathway in the brain;
neurotransmission, neurotransmitters (including dopamine), and reuptake transporters that
recycle neurotransmitters for future use;
FIGURE 2
In vivo microdialysis and HPLC.
◆
◆
◆
◆
reward pathways using laboratory animals;
“hijacking” of the pathways by drugs such as
cocaine;
scientific investigations injecting drugs directly
into the brain to verify the location of reward
pathways; and
the mechanisms through which drugs like cocaine increase dopamine transmission.
Explain that after drugs make dopamine bombard
its receptors for long periods of time, the body is no
longer able to transmit dopamine normally. When the
body receives unnatural stimulation from drugs, it can
respond by down-regulating its own neurotransmission in the same system by decreasing neurotransmitter synthesis or the number of post-synaptic receptors.
Then, when drugs are no longer present, the system is
depressed. This can contribute to psychological depression, anxiety, and other psychological disorders associated with drug use and abuse.
Use the information in the following section to produce additional slides or outline notes to help explain
in vivo microdialysis and HPLC; ask students again to
take notes and later answer the Explorer Guide questions (Figure 1).
In vivo microdialysis and HPLC
In vivo microdialysis
One method used to analyze the effects of natural rewards and drugs on the brain is in vivo microdialysis.
This technique allows scientists to sample chemicals
in the extracellular fluid around cells in the brains
of laboratory animals (Figure 2). A microdialysis
probe is surgically implanted into a brain region of
interest (e.g., the nucleus accumbens). The tip of the
probe contains an inlet tube and an outlet tube surrounded by a semipermeable cellulose membrane.
An experimenter pumps artificial cerebrospinal fluid
(aCSF)—containing water, sugar, and salt solution
that is pH-balanced—through the inlet tube of the
probe in the brain.
Due to laws of diffusion, chemicals more concentrated in the brain than in the aCSF cross the probe’s
membrane into the aCSF. A collection vial gathers
aCSF from the outlet tube. The aCSF now contains
brain chemicals and is called a microdialysis sample (or
sample). The interesting brain chemicals in the sample
are called analytes. Samples are often collected every 10
or 20 minutes in a volume of 10–20 µl. Microdialysis
samples can be collected while an animal carries out
normal behaviors, such as eating food, drinking water,
or receiving a drug injection in an experiment.
Because neurotransmission occurs via chemical messengers (called neurotransmitters) released into the synapse,
chemical messengers can diffuse away from the synapse
January 2007
35
The main components of an
HPLC system are the mobile phase
The Human HPLC Column.
(or liquid phase) and the stationary
phase (or solid phase). The mobile
(Figure modified from Frantz 2004.)
phase serves as a solvent in which
analytes from the microdialysis
sample are dissolved. The pH and
chemical characteristics of the mobile phase control the ionic state of
the analyte molecules. The liquid
mixture is pumped through a steel
tube (HPLC column) packed with
very small (3–10 µm diameter)
silica (sand) particles. The solid
phase is made up of an immobile
material bonded to the surface of
the silica particles.
A common form of HPLC is
Reversed-Phase Chromatography
Sample Chromograph
in which the mobile phase is a
polar solution of water with an
organic chemical (e.g., methanol
or acetonitrile) and the solid phase
is made nonpolar because nonpolar chemicals such as hydrocarbon
chains (e.g., hydrophobic alkyls, Retention time (s)
CH2-CH2-CH2-CH3) are attached
to the surface of the silica particles. A hydroxyl group attached
to a silicate atom is often called
a silanol. [Note: This method is
called reversed-phase because traditionally the mobile phase was
nonpolar and the stationary phase
was polar.]
Using an injector, analytes from a liquid sample (e.g.,
and into an implanted microdialysis probe. Therefore, a
chemicals in a microdialysis sample) are injected onto
microdialysis sample reveals information about neurothe column, mixed with the mobile phase solution, and
transmission in the brain. For example, if dopamine is
pumped across the column at high pressure. [Note:
released at high rates from its synapse, some dopamine
HPLC was previously known as High Pressure Liquid
will diffuse away into the nearby microdialysis probe and
Chromatography.] The following Human HPLC Colinto the sample. High concentrations of dopamine in the
umn activity models the process of chemical separation.
sample reflect high levels of dopamine release from neu[Note: Advanced learners and instructors interested in
rons (brain cells) in the brain.
more detail on HPLC can view the online book HPLC
HPLC
for Pharmaceutical Scientists (see “On the web”).]
HPLC is a method of chemical separation that is often
The Human HPLC Column
coupled with microdialysis. Microdialysis samples are
Everyone can participate in this choreography activanalyzed with HPLC to determine what types and
ity, which has students model and act out the process of
how many analyte molecules are present. By comparchemical separation. Recruit nearly everyone to represent
ing sequential samples over time while an animal is
Solid-Phase silica particles in an HPLC column. Hold
behaving in a certain manner (e.g., drinking water),
about 7 to 10 students back to serve in the other roles. In
HPLC can reveal which analytes, such as neurotranssmaller classes, recruit at least 6 to 8 volunteer students
mitters, are increased or decreased due to the animal
to make up the Human HPLC Column. Line those stubehavior. Alternatively, HPLC can reveal the brain
dents up facing one another at the front of the class or
concentration of injected drugs.
(number of students)
Concentration of analyte
FIGURE 3
36
The Science Teacher
FIGURE 4
Sample data collection and analysis tables.
A.
Analyte
Retention time(s)
Dopamine 1
6
Dopamine 2
7
Cocaine 1
9
Cocaine 2
10
Cocaine 3
9.5
Benzoylecgonine 1
12
Benzoylecgonine 2
12
Analyte
Retention time(s)
Dopamine 1
6
Dopamine 2
7
Cocaine 1
9
Cocaine 2
10
Cocaine 3
9.5
Benzoylecgonine 1
12
Benzoylecgonine 2
12
B.
Number of analyte molecules
(amount of analyte)
Average retention time(s) by
type of analyte
2
6.5
3
9.5
2
12
possibly in a long hallway. Have students wave their arms
as though their arms were hydrocarbons attached to the
silica beads (Figure 3). Their fingertips should be about
15 cm from touching across the column, so move lines
closer or farther apart as necessary.
These students represent the solid phase silica beads
with hydrocarbon chains packed into the HPLC column.
The silica beads interact differently with various analytes
in a liquid sample due to characteristics of the analytes,
such as hydrophobicity. [Note: More hydrophobic components of an analyte will interact longer with the nonpolar
solid phase hydrocarbon chains.] Students will model
such intermolecular attractions using arm waving and
handshakes, as described next.
Allow the HPLC column Solid-Phase volunteers to
rest their arms but pay attention to instructions for the
other volunteers. Recruit at least four students to serve
as Analyte molecules in the microdialysis samples from
the extracellular fluid; assign them roles of Dopamine,
Cocaine, or Benzoylecgonine (a breakdown product, or
metabolite, of cocaine; pronounced BENZ-oil-ECK-oggneen). In order to incorporate math and replication in
data collection, it is best to have at least two students represent each type of analyte; the more, the better. Gather
the Analytes at the top of the HPLC Column. Instruct
the Analyte students that when the Injector volunteer
starts the sample (described next), Analytes will model
the process of chemical separation as follows.
◆
◆
◆
◆
Students representing Dopamine progress down
the column freely by walking (not running) between the columns of Solid-Phase volunteers.
Students representing Cocaine progress down
the column waving their own arms, causing brief
contact with the Solid-Phase volunteers who are
still waving their arms. (Instruct students not to
punch one another!)
Students representing Benzoylecgonine progress down the column shaking hands with
each and every Solid-Phase volunteer, simulating intermolecular attractions between analyte
and solid phase.
Predictably, Dopamine will “run” the column
faster than Cocaine, which will run faster than
Benzoylecgonine, thereby separating the chemicals by retention time. To decrease data variability, a baseline speed for Analytes walking down
the column may be agreed upon and practiced
without the arm movements before beginning.
Instruct the Solid-Phase and Analyte volunteers to
wait while paying attention to instructions for the last
January 2007
37
three volunteers. Recruit one Injector volunteer to initiate
sample injection by saying “Go!” to each Analyte lined up
at the head of the column. Recruit a Detector volunteer
to track retention time, which is the number of seconds
required for each molecule to progress down the column.
The Detector should start the stopwatch when the Injector says go, and should stop the stopwatch and announce
the arrival of the molecule at the end of the column by
saying “Stop!” when the Analyte reaches the end. [Note:
The Detector should be positioned at the end of the column.] Recruit a Chart Recorder to record the retention
time on a data collection table on the board (see Figure
4a, p. 37 for an example).
Once all students understand their assignments, run
the sample on the column. Allow one Analyte molecule at
a time to walk down the HPLC Column. It is best to run
all Analytes of one type first, then the second type, and so
on, simply because in a real column, the analytes of one
type would gradually bunch together and run off the end
of the column at approximately the same time. However,
if you have a small number of participants, you can run
each Analyte volunteer through the column several times
to collect more data points for analysis.
After all data are collected on the table (Figure 4a),
students may return to their seats to begin data analysis.
Either individually or as a group, students count the
number of analyte molecules (or replications of analyte
molecule retention times) and place them in a column on
an expanded data analysis table (see Figure 4b, p. 37, for
an example). Next students calculate the average retention time by type of analyte and record that in the last
column. Finally, students transfer the data to a graph on
their Explorer Guides (see graph in Figure 3, p. 36). Consider this graph to be a chromatogram representing data
collected from a microdialysis probe collecting analytes
from the nucleus accumbens of the rat brain over 10–20
minutes after an injection of cocaine to the rat.
As a class, review key ideas on the chromatogram.
First, the Y-axis represents the amount of analyte per
sample (concentration), whereas the X-axis represents
retention time (in seconds). Second, data on the concentration of analyte reveals how much of each analyte was
present in the sample. It is the retention time that identifies the type of analyte. Third, cocaine increases dopamine
in the microdialysis sample because it blocks dopamine
reuptake from the synapse, leaving more dopamine available to diffuse across the microdialysis membrane into the
aCSF. Cocaine increases in the brain because it was injected into the animal and circulates in the blood stream
throughout the body, including the brain. Benzoylecgonine increases because metabolic enzymes in the body
break down cocaine into several metabolites for excretion
from the body. Benzoylecgonine is one of the metabolites,
formed by the hydrolysis of cocaine by the liver and excreted in the urine of cocaine users.
38
The Science Teacher
Finally, in a true experiment using HPLC to quantify brain chemicals, we would run standards of several known concentrations of individual known analytes and graph results on a chromatogram. Then, we
would compare the peak heights and retention times
from the sample chromatogram with the peak heights
and retention times from the standard chromatograms,
to determine the amounts and types of analytes in the
microdialysis sample.
With regard to troubleshooting a messy graph, if
student analyte peaks are almost on top of each other
(indicating very similar retention time for different
analytes), then students have encountered a common
problem in HPLC. In the laboratory, researchers
would change the length of the hydrocarbon chain in
the solid phase, or the pH or concentration of organic
solvent in the mobile phase, to cause one analyte to
move faster or slower than the other down the column,
thereby fully separating the peaks for each analyte. In
the student activity, modifications in contact time can
improve the results; for example, the Benzoylecgonine
students might be instructed to shake hands twice with
each of the Solid-Phase volunteers.
If these concepts come easily to some students,
ask them to consider how the data would change
under different conditions. Provide extra copies of
blank chromatograms and ask them to regraph the
chromatogram to reflect the following differences.
[Note: Providing unlabeled graphs requires students
to generate axis labels, peak labels, and a graph title,
increasing the challenge.]
◆
◆
◆
Administration of a higher dose of cocaine. (This
would elevate brain levels of dopamine, cocaine,
and benzoylecgonine.)
Metabolic tolerance due to repeated exposure
to the same dose of cocaine. (This would lower
levels of dopamine and cocaine, but elevate benzoylecgonine due to faster metabolism.)
Sensitization of the pharmacological effect of cocaine after repeated exposure to the same dose of
cocaine. (This would elevate dopamine, but leave
cocaine and benzoylecgonine unchanged.)
Assessment
Use students’ worksheets and graphs to assess comprehension. We recommend allowing students to work in
pairs; it increases the verbal use of relevant vocabulary
and encourages peer instruction. Successful completion
of worksheets could lead to the following extension activities as homework assignments or group projects:
◆
See what happens if all analytes are injected onto
the Human HPLC Column at the same time.
(You might need to widen the column.) This
◆
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◆
◆
◆
◆
◆
model would be more like real sample injections.
Do the different types of analytes separate from
one another and reach the end of the column at
different times?
Investigate other mechanisms of chemical separation in HPLC, such as size exclusion. Let students design ways to model size exclusion chromatography in the classroom.
Design an experiment using HPLC to assess the
chemical makeup of any solution.
Calculate the flow rate (in µL/min) required
to collect a 10 µL sample in 10 minutes. Then
recalculate for the flow rate required to collect a
10 µL sample in 6 minutes, and so on.
Create a slide show explaining the intracellular cyclic adenosine monophosphate (cAMP)
pathways through which dopamine can affect
cellular activity.
Record in a journal the long-term effects of psychostimulant drug intake, including psychological depression, anxiety, and other psychological
disorders that accompany low levels of dopamine
activity in the brain.
Record in a journal the difference between basic
research in neuroscience (e.g., in vivo microdialysis with HPLC) and clinical applications
of neuroscience (e.g., development and testing
of behavioral and pharmacological drug abuse
treatment programs). What types of information
does each contribute to our understanding of
drug use and abuse?
Debate ethical concerns regarding the use of humans and other animals in research.
Extensions and modifications of the lesson plan facilitate integration across the curriculum from biomedical technology (e.g., focus on in vivo microdialysis and
HPLC) to biology (e.g., brain anatomy, neurotransmission, extracellular fluid composition), psychology (e.g.,
drug effects on behavior, drug addiction, depression,
anxiety), chemistry (e.g., diffusion, polarity, pH), math
(e.g., averaging data points, graphing, calculating concentration or flow rate), and health (e.g., societal impact
Correlation with Standards.
This activity was designed to correlate with the following
9–12 Content Standards (NRC 1996):
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◆
◆
A: Science as Inquiry (p. 121)
B: Physical Science (p. 123)
C: Life Science (p. 127)
E: Science and Technology (p. 135)
F: Science in Personal and Social Perspective (p. 138)
of drug addiction, other diseases or risks associated with
drug abuse).
Follow-up discussions should drive home the important message that if drugs such as cocaine cause dopamine
to bombard its receptors at unnaturally high levels over
unnaturally long periods of time, then eventually the
body responds by down-regulating its own dopamine activity. Such dysregulation of dopamine transmission can
contribute to psychological depression, anxiety, and other
psychological disorders that are associated with drug use
and abuse. Active exploration of such a relevant topic
may spur maturation of analytical-reasoning skills while
sparking student enthusiasm for science. ■
Kyle Frantz ([email protected]) is an assistant professor in the Department of Biology at Georgia State University in Atlanta, Georgia.
Acknowledgments
The author would like to thank David Parlier for thoughtful review
and revision of this manuscript, as well as all students who have enthusiastically participated in this module.
References
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Sciences 1021: 323, 371–375.
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On the web
Slide Teaching Packets (www.nida.nih.gov/pubs/Teaching)
Substance Abuse and Mental Health Services Administration (www.
samhsa.gov)
Center for Behavioral Neuroscience (www.cbn-atl.org)
Neuroscience for Kids (http://faculty.washington.edu/chudler/
neurok.html)
HPLC for Pharmaceutical Scientists book (http://hplc.chem.shu.edu/
HPLC/index.html)
January 2007
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